Methods and oligonucleotides for the detection of Salmonella sp., E. coli O157:H7, and Listeria monocytogenes

ABSTRACT

A method for detecting a Salmonella species,  E. coli  O157:H7, or  Listeria monocytogenes  is disclosed. The method involves amplifying a genomic nucleotide sequence of a corresponding species and detecting the amplification product. Various primers and probes that can be used in the method are also disclosed. In one embodiment, the amplification step of the method is accomplished by real-time PCR and the amplification product is detected by fluorescence resonance energy transfer using a pair of labeled polynucleotides.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. application SerialNo. 60/300,199, filed on Jun. 22, 2001, U.S. application Serial No.60/373,588, filed on Apr. 18, 2002, and U.S. application Serial No.60/373,589, filed on Apr. 18, 2002.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] None.

BACKGROUND OF THE INVENTION

[0003] Federal and state health and safety standards mandate thatindustrial food service companies and manufacturing facilities performroutine testing for common bacteria, such as Salmonella species, E. coliO157:H7, and Listeria monocytogenes, that cause food-borne illnesses. Asa safety precaution, companies are required to perform testing on eachbatch or lot of food prior to the food reaching the public. Severalmethods are currently available for industrial testing of bacteria inthe food service industry.

[0004] However, there are currently severe limitations on the testsavailable to the industry. Present methods utilized as industrystandards require 2-5 days to perform. For example, the most widely usedmethods for detection of Salmonella employ a pre-enrichment (day 1), aselective enrichment (day 2), and a final enrichment followed by animmunoassay requiring 10⁵ organisms (day 3); the most widely usedmethods of detection of E. coli O157:H7 employ a selective enrichment(8-28 hours) and an immunoassay requiring 10⁵ organisms; the most widelyused methods of detection of Listeria monocytogenes employ apre-enrichment (26-30 hours), an enrichment (22-26 hours), and animmunoassay requiring 10⁵ organisms. For the detection of E. coliO157:H7 and Listeria monocytogenes, all samples that are suspected aspositive by the immunoassay must be confirmed by culture methods (1-3days for E. coli O157:H7 and 4-5 days for Listeria monocytogenes). Thus,in many cases, the food suppliers must wait days for test results beforeshipping their already manufactured products. As a result, the companymay lose profits from a reduced shelf life and the wait also increasesthe potential for food spoilage.

[0005] In addition, using methods now available in the art, the organismneeds to be cultured to a concentration of at least 10⁵/ml to bedetected. Because the margin of error in detectability of the bacteriais high, false negative tests may result and a food poisoning outbreakmay occur. The company is then forced to recall product that has alreadyreached the consumer. This places the public at a great health risk. Themanufacturer or producer is also forced to bear the costs of recall, andis at a risk for lawsuit or government mandated shutdown of productionfacilities.

[0006] Thus, there is a need for an inexpensive testing technology thatprovides a rapid turn-around time, and a high degree of accuracy andreproducibility, which will result in safer food manufacturing andpreparation. Additionally, there is a need for a method that keeps pacewith new manufacturing processes. Polymerase chain reaction (“PCR”)testing technology for food-borne pathogenic bacteria facilitates rapidand accurate testing for the manufacturers.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention provides a method for detecting aSalmonella species, E. coli O157:H7, or Listeria monocytogenes. Themethod involves amplifying a genomic nucleotide sequence of acorresponding species and detecting the amplification product. Thepresent invention also encompasses primers and probes that can be usedin the method. The primers and probes can be provided in a detectionkit.

[0008] In one embodiment, the amplification step of the method of thepresent invention is accomplished by real-time PCR and the amplificationproduct is detected by fluorescence resonance energy transfer using apair of labeled oligonucleotides.

[0009] It is a feature of the present invention that the genomic regionfrom which a nucleotide sequence is amplified is involved in bacterialvirulence.

[0010] It is an advantage of the present invention that the method ofbacteria detection is sensitive.

[0011] It is another advantage of the present invention that the methodof bacteria detection is fast.

[0012] Other objects, advantages, and features of the present inventionwill become apparent from the following detailed description of theinvention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0013] Not applicable.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to the detection of bacterialpathogens in food or other materials with much greater sensitivity andspeed than was heretofore possible. Primers have been identified whichpermit a rapid and sensitive type of polymerase chain reaction (PCR) toamplify target DNA if, and only if, one of the target pathogens ispresent in a sample. Probes are also identified which will bind to theamplified DNA products produced again if, and only if, the organism ispresent. The method has been implemented for Salmonella, E. coliO157:H7, and Listeria monocytogenes.

[0015] An used herein, an “isolated nucleic acid” is a nucleic acidwhich may or may not be identical to that of a naturally occurringnucleic acid but which is isolated from a living host organism. When“isolated nucleic acid” is used to describe a primer or a probe, thenucleic acid is not identical to the structure of a naturally occurringnucleic acid spanning at least the length of a gene.

[0016] In one aspect, the present invention relates to nucleic acidsthat can be used as primers to amplify a genomic fragment isolated fromSalmonella species, E. coli O157:H7 or Listeria monocytogenes to detectthe corresponding species. Such a nucleic acid has a nucleotide sequencecontaining at least 12 consecutive nucleotides of SEQ ID NO:1 (5′ primerfor Salmonella species), SEQ ID NO:2 (3′ primer for Salmonella species),SEQ ID NO:5 (5′ primer for E. coli O157:H7), SEQ ID NO:6 (3′ primer forE. coli O157:H7), SEQ ID NO:9 (5′ primer for Listeria monocytogenes), orSEQ ID NO:10 (3′ primer for Listeria monocytogenes). Preferably, thenucleic acid has a sequence that contains at least 15 or 18 consecutivenucleotides, and most preferably the fill length, of theabove-identified sequences.

[0017] In another aspect, the present invention relates to labelednucleic acids that can act as probes to facilitate the detection of anamplification product of a Salmonella species, E. coli O157:H7 orListeria monocytogenes, obtained using the primers described above. Thelabeled nucleic acid probes work in pairs. One probe in each pair islabeled at the 3′ end and the other probe is labeled at the 5′ end. Eachprobe pair hybridize to the same strand of the amplification product.When hybridized to the amplification product, the 3′ end nucleotide ofthe 3′ end labeled nucleic acid probe and the 5′ end nucleotide of the5′ end labeled nucleic acid probe are less than six nucleotides apart sothat energy transfer occurs between the two labels resulting in anemission intensity change of at least one of the labels. The emissionintensity change indicates the presence of the amplification product.

[0018] The labeled nucleic acid probes in each pair have nucleotidesequences containing at least 12 consecutive nucleotides of SEQ ID NO:13(for Salmonella species), the complement of SEQ ID NO:13 (for Salmonellaspecies), SEQ ID NO:14 (for E. coli O157:H7), the complement of SEQ IDNO:14 (for E. coli O157:H7), SEQ ID NO:15 (for Listeria monocytogenes),or the complement of SEQ ID NO:15 (for Listeria monocytogenes).Preferably, the labeled nucleic acids in each probe pair have nucleotidesequences containing at least 15 or 18 nucleotides of theabove-identified sequences. Most preferably, the labeled nucleic acidsin each pair have the following pair of nucleotide sequences: SEQ IDNO:3 and SEQ ID NO:4 (for Salmonella species), the complement of SEQ IDNO:3 and the complement of SEQ ID NO:4 (for Salmonella species), SEQ IDNO:7 and SEQ ID NO:8 (for E. coli O157:H7), the complement of SEQ IDNO:7 and the complement of SEQ ID NO:8 (for E. coli O157:H7), SEQ IDNO:11 and SEQ ID NO:12 (for Listeria monocytogenes), and the complementof SEQ ID NO:11 and the complement of SEQ ID NO:12 (for Listeriamonocytogenes).

[0019] Any pair of labeling molecules that can undergo energy transferwhen located close to each other (less than 6 nucleotides apart on anucleotide sequence) to cause a change in emission intensity in at leastone of the labeling molecules can be used to make the labeled nucleicacids described above. An example of a labeling molecule for one nucleicacid in a pair includes, but are not limited to, fluorescein. Examplesof labeling molecules for the other nucleic acid in the pair include butare not limited to LC RED 640 (Roche Lightcycler), LC RED 705 (RocheLightcycler).

[0020] In another aspect, the present invention relates to a kit fordetecting at least one of a Salmonella species, E. coli O157:H7 andListeria monocytogenes. The kit contains a pair of nucleic acid primersand a pair of labeled nucleic acids, as described above, for one, two orall three of the above species. Other reagents for the amplification ofa target DNA and the detection of the amplification product can also beincluded in the kit. The kit may also include positive and negativecontrols for the above species. The positive control can be any samplethat contains a target DNA to be amplified, including the bacteriathemselves, at an amount over the detection limit. The negative controlis a sample that does not contain the target DNA to be amplified.

[0021] In another aspect, the present invention relates to an isolatednucleic acid the amplification of which allows detection of a Salmonellaspecies, E. coli O157:H7 or Listeria monocytogenes. Examples of suchnucleic acids include those that contain SEQ ID NO:13, SEQ ID NO:14 orSEQ ID NO:15.

[0022] In still another aspect, the present invention relates to amethod for detecting a Salmonella species, E. coli O157:H7, or Listeriamonocytogenes. The method involves amplifying a fragment of the genomicDNA specific to the above species and detecting the amplificationproduct. Unique sequences that can be used to identify a Salmonellaspecies, E. coli O157:H7, and Listeria monocytogenes include nucleotide2314 to nucleotide 2047 (nucleotide 9 to nucleotide 243 of SEQ ID NO:13)of the sipB-sipC region of the Salmonella genome (GenBank Accession No.U25631), nucleotide 1185 to nucleotide 1532 (nucleotide 7 to nucleotide354 of SEQ ID NO:14) of the eae gene of E. coli O157:H7 (GenBankAccession No. AF081182), and nucleotide 2995 to nucleotide 3196(nucleotide 9 to nucleotide 210 of SEQ ID NO:15) of the internalinoperon of Listeria monocytogenes (GenBank Accession No. AJ012346). Anygenomic fragments that contain the above sequences can be amplified fordetecting the above species. Given what is disclosed herein, a skilledartisan knows how to amplify a fragment that contains one of the abovespecific sequences and then detect the presence of an amplificationproduct that contains the sequence. Examples of the primers that can beused in the method of present invention are described above.

[0023] The genomic sequences amplified and detected with the method ofthe present invention are from genomic regions that are involved inbacterial virulence. The sip proteins of the Salmonella species and theinternalin proteins of Listeria monocytogenes are required for cellinvasion; the EAE proteins of E. coli O157:H7 are required for celleffacement and attachment. Thus, the method of the present inventiondetects bacteria that harbor virulent traits. Nonpathogenic strains ofthese species are not meant to be detected using this technique.

[0024] It is understood that the species specific sequences actuallyamplified in performing the method of the present invention may varysomewhat from the sequences described above. The variations may becaused by sequencing errors, strain-specific variations or some otherreasons. The method of the present invention intends to encompass thesevariations.

[0025] In a specific embodiment, a fragment of genomic DNA specific to aspecies is amplified by real-time PCR and the amplification product isdetected by fluorescence resonance energy transfer (FRET) using labelednucleic acids described above as internal hybirdization probes. In thisembodiment, internal hybridization probes are included in the PCRreaction mixture so that product detection occurs as the product isformed, further reducing post-PCR processing time. Roche Lightcycler PCRinstrument (U.S. Pat. No. 6,174,670) or other real-time PCR instrumentscan be used in this embodiment of the invention. PCR amplification ofDNA allows for the increase in sensitivity to less than 10¹ organisms incomparison to 10⁵ organisms in standard immuno-detection methodspresently used. Real-time PCR amplification and detection can reducetotal assay time so that test results can be obtained within 12 hours.

[0026] The invention will be more fully understood upon consideration ofthe following non-limiting examples.

EXAMPLE 1 Detection of Salmonella species

[0027] A sample of the food product was weighed out and mixed withBuffered Peptone Water. The ratio of the food product to BufferedPeptone Water was 25 to 225 (grams to mls). The mixture was thenmechanically homogenized and incubated at 35+/−2° C. After six hours ofincubation, 15 ml of mixture was removed and centrifuged at 2,500× g for10 minutes. The supernatant was discarded and the pellet was resuspendedin 200 ml of TE. The DNA was then extracted from the bacteria usingeither the Qiagen QIAamp DNA mini kit (Qiagen Inc., Valencia, Calif.) orBiotecon foodproof® extraction kit (Potsdam, Germany).

[0028] Next, PCR amplification and detection of amplification productwere performed. The following oligonucleotides were designed to providefor the PCR amplification of a 250 bp product spanning from base 2305 tobase 2555 of the sipB-sipC region of the Salmonella genome (GenBankAccession #U25631): forward 5′-ACAGCAAAATGCGGATGCTT-3′ (SEQ ID NO:1) andreverse 5′-GCGCGCTCAGTGTAGGACTC-3′ (SEQ ID NO:2).

[0029] In addition, internal hybridization probes were designed to allowfor detection of the PCR product by fluorescence resonance energytransfer within the Roche Lightcycler. The sequence and modifications ofthe probes were: upstream5′-GCAATCCGTTAGCGCTAAAGATATTCTGAATAGT-Fluorescein-3′ (SEQ ID NO:3) anddownstream 5′-LC RED640TTGGTATTAGCAGCAGTAAAGTCAGTGACCTGG-Phos-3′ (SEQ IDNO:4). These probes were designed to anneal to the upper strand frompositions 2464-2497 (upstream) and 2499-2531 (downstream). PCRoptimization was then carried out to allow for rapid real-timeamplification and detection in the Roche Lightcycler PCR instrument(U.S. Pat. No. 6,174,670). PCR amplification of DNA led to an increasein sensitivity to less than 10¹ organisms in comparison to 10⁵ organismsin standard prior art immuno detection methods. These hybridizationprobes provided a high degree of specificity and accurate detection ofSalmonella isolates. No false positives were observed.

[0030] This test methodology detected Salmonella at the lowpre-enrichment concentration range of 10⁰ organisms/ml-10¹ organisms/mlby amplification of DNA using oligonucleotides. Utilizing the RocheLightcycler, which completed cycles in about 30 minutes, instead ofhours or overnight, as in older thermocyclers, allowed test results tobe obtained within 12 hours.

EXAMPLE 2 Detection of E. coli O157:H7

[0031] A sample of the food product was weighed out and mixed withmodified Trypticase Soy Broth. The ratio of the food product to modifiedTrypticase Soy Broth was 25 to 225 (grams to mls). The mixture was thenmechanically homogenized and incubated at 35+/−2° C. After six hours ofincubation, 15 ml of mixture was removed and centrifuged at 2,500× g for10 minutes. The supernatant was discarded and the pellet wasre-suspended in 200 ml of TE. The DNA was then extracted from there-suspended bacteria using either the Qiagen QIAamp DNA mini kit(Qiagen Inc., Valencia, Calif.) or Biotecon foodproof® extraction kit(Potsdam, Germany).

[0032] Next PCR amplification and detection of PCR amplification productwere performed. The following oligonucleotides were designed to providefor the PCR amplification of a 361 bp product spanning from base 1179 tobase 1539 of the eae gene of the E. coli O157:H7 genome (GenBankAccession #AF081182): forward 5′-TGGTACGGGTAATGAAAA-3′ (SEQ ID NO:5) andreverse 5′-AATAGCCTGGTAGTCTTGT-3′ (SEQ ID NO:6).

[0033] In addition, internal hybridization probes were designed fordetection of the PCR product by fluorescence resonance energy transferwithin the Roche Lightcycler. The sequence and modifications of theprobes were: upstream 5′-CGCAGTCAGGGCGGTCAGA-Fluorescein-3′ (SEQ IDNO:7) and downstream 5′-LC RED640TCAGCATAGCGGAAGCCAAA-Phos-3′ (SEQ IDNO:8). These probes were designed to anneal to the upper strand frompositions 1477-1495 (upstream) and 1497-1516 (downstream). PCRoptimization was then carried out to allow for rapid real-timeamplification and detection in the Roche Lightcycler PCR instrument(U.S. Pat. No. 6,174,670) or other real-time PCR instrument. PCRamplification of DNA led to an increase in sensitivity to less than 10¹organisms in comparison to 10⁵ organisms in standard prior art immunodetection methods. These hybridization probes provided a high degree ofspecificity and accurate detection of E. coli O157:H7 isolates. No falsepositives were observed.

[0034] Utilizing the Roche Lightcycler, which completed cycles in about30 minutes, instead of hours or overnight, as in older thermocyclers,allowed test results to be obtained within 12 hours.

EXAMPLE 3 Detection of Listeria monocytogenes

[0035] Two hundred and twenty five ml of Fraser broth was added to asample of 25 grams of the food product. The mixture was then stomachedand incubated at 30° C. After eight hours of incubation, 15 ml ofmixture was removed and centrifuged at 2,500× g for 10 minutes. Thesupernatant was discarded and the pellet was resuspended in 200 ml TE.The DNA was then extracted from the resuspended bacteria using eitherthe Qiagen QIAamp DNA mini kit (Qiagen Inc., Valencia, Calif.) orBiotecon foodproof® extraction kit (Potsdam, Germany).

[0036] Next, PCR amplification and detection of PCR amplificationproduct were performed. The following oligonucleotides were designed toprovide for the PCR amplification of a 217 bp product spanning from base2987 to base 3203 of the internalin operon of the Listeria monocytogenesgenome: forward 5′-ATTTAGTGGAACCGTGACGC-3′ (SEQ ID NO:9) and reverse5′-GATGTCATTTGTCGGCATT-3′ (SEQ ID NO:10).

[0037] In addition, internal hybridization probes were designed to allowfor detection of the PCR product by fluorescence resonance energytransfer within the Roche Lightcycler. The sequence and modifications ofthe probes were upstream 5′-AGCTAAGCCCGTAAAAGAAGGT-Fluorescein-3′ (SEQID NO:11) and downstream 5′-LC RED640-ACACATTTGTTGGTTGGTTTGATGCC-Phos-3′(SEQ ID NO:12). These probes were designed to anneal to the upper strandfrom positions 3098-3119 (upstream) and 3121-3146 (downstream). PCRoptimization was then carried out to allow for rapid real-timeamplification and detection in the Roche Lightcycler PCR instrument(U.S. Pat. No. 6,174,670) or other real-time PCR instrument. Thesehybridization probes provided a high degree of specificity and accuratedetection of Listeria monocytogenes isolates. No false positives wereobserved.

[0038] Utilizing the Roche Lightcycler, which completed cycles in about30 minutes, instead of hours or overnight, as in older thermocyclers,allowed test results to be obtained within 12 hours.

[0039] The present invention is not intended to be limited to theforegoing examples, but encompasses all such modifications andvariations as come within the scope of the appended claims.

1 15 1 20 DNA Artificial Sequence Description of Artificial SequencePCRprimer for Salmonella sp. 1 acagcaaaat gcggatgctt 20 2 20 DNA ArtificialSequence Description of Artificial SequencePCR primer for Salmonella sp.2 gcgcgctcag tgtaggactc 20 3 34 DNA Artificial Sequence Description ofArtificial SequenceDetection probe for Salmonella sp. 3 gcaatccgttagcgctaaag atattctgaa tagt 34 4 33 DNA Artificial Sequence Descriptionof Artificial SequenceDetection probe for Salmonella sp. 4 ttggtattagcagcagtaaa gtcagtgacc tgg 33 5 18 DNA Artificial Sequence Description ofArtificial SequencePCR primer for E. coli O157H7 5 tggtacgggt aatgaaaa18 6 19 DNA Artificial Sequence Description of Artificial SequencePCRprimer for E. coli O157H7 6 aatagcctgg tagtcttgt 19 7 19 DNA ArtificialSequence Description of Artificial SequenceDetection probe for E. coliO157H7 7 cgcagtcagg gcggtcaga 19 8 20 DNA Artificial SequenceDescription of Artificial SequenceDetection probe for E. coli O157H7 8tcagcatagc ggaagccaaa 20 9 20 DNA Artificial Sequence Description ofArtificial SequencePCR primer for Listeria monocytogenes 9 atttagtggaaccgtgacgc 20 10 19 DNA Artificial Sequence Description of ArtificialSequencePCR primer for Listeria monocytogenes 10 gatgtcattt gtcggcatt 1911 22 DNA Artificial Sequence Description of ArtificialSequenceDetection probe for Listeria monocytogenes 11 agctaagcccgtaaaagaag gt 22 12 26 DNA Artificial Sequence Description of ArtificialSequenceDetection probe for Listeria monocytogenes 12 acacatttgttggttggttt gatgcc 26 13 251 DNA Salmonella sp. misc_feature (1)..(251)Fragment of sipB-sipC region 13 acagcaaaat gcggatgctt cgcgttttattctgcgccag agtcgcgcat aaaaactgcc 60 aaaataaagg gagaaaaata tgttaattagtaatgtggga ataaatcccg ccgcttattt 120 aaataatcat tctgttgaga atagttcacagacagcttcg caatccgtta gcgctaaaga 180 tattctgaat agtattggta ttagcagcagtaaagtcagt gacctggggt tgagtcctac 240 actgagcgcg c 251 14 361 DNA E. coliO157H7 misc_feature (1)..(361) Fragment of eae gene 14 tggtacgggtaatgaaaatg atctccttta ctcaatgcag ttccgttatc agtttgataa 60 atcgtggtctcagcaaattg aaccacagta tgttaacgag ttaagaacat tatcaggcag 120 ccgttacgatctggttcagc gtaataacaa tattattctg gagtacaaga agcaggatat 180 tctttctctgaatattccgc atgatattaa tggtactgaa cacagtacgc agaagattca 240 gttgatcgttaagagcaaat acggtctgga tcgtatcgtc tgggatgata gtgcattacg 300 cagtcagggcggtcagattc agcatagcgg aagccaaagc gcacaagact accaggctat 360 t 361 15 217DNA Listeria monocytogenes misc_feature (1)..(217) Fragment ofinternalin operon 15 atttagtgga accgtgacgc agccacttaa ggcaatttttaatgttaagt ttcatgtgga 60 cggcaaagaa acaaccaaag aagtggaagc tgggaatttattgactgaac cagctaagcc 120 cgtaaaagaa ggtcacacat ttgttggttg gtttgatgcccaaacaggcg gaactaaatg 180 gaatttcagt acggataaaa tgccgacaaa tgacatc 217

We claim:
 1. An isolated nucleic acid comprising at least 12 consecutivenucleotides of a nucleotide sequence selected from SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO:10.
 2. Theisolated nucleic acid of claim 1, wherein the nucleic acid comprises atleast 15 consecutive nucleotides of the nucleotide sequence.
 3. Theisolated nucleic acid of claim 1, wherein the nucleic acid comprises atleast 18 consecutive nucleotides of the nucleotide sequence.
 4. Theisolated nucleic acid of claim 1, wherein the nucleic acid comprises anucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO:10.
 5. A pair of labelednucleic acids: the first labeled nucleic acid comprises at least 12consecutive nucleotides of a nucleotide sequence selected from SEQ IDNO:13, the complement of SEQ ID NO:13, SEQ ID NO:14, the complement ofSEQ ID NO:14, SEQ ID NO:15, and the complement of SEQ ID NO:15 on the 3′end region, and labeling molecule one at the 3′ end; and the secondlabeled nucleic acid comprises at least 12 consecutive nucleotides ofthe same nucleotide sequence on the 5′ end region, and labeling moleculetwo at the 5′ end; wherein when the two labeled nucleic acids hybridizeto the complement of the nucleotide sequence, the nucleotide at the 3′end of the first labeled nucleic acid and the nucleotide at the 5′ endof the second labeled nucleic acid are separated by less than sixnucleotides and at least one of labeling molecule one and labelingmolecule two changes emission intensity at a wavelength due to energytransfer between the two labeling molecules.
 6. The pair of labelednucleic acids of claim 5, wherein the first labeled nucleic acidcomprises at least 15 consecutive nucleotides of the nucleotide sequenceand the second labeled nucleic acid comprises at least 15 consecutivenucleotides of the same nucleotide sequence.
 7. The pair of labelednucleic acids of claim 5, wherein labeling molecule one is fluorescein,and labeling molecule two is selected from LC RED 640 and LC RED
 705. 8.The pair of labeled nucleic acids of claim 5, wherein the nucleic acidsequences of each pair are selected from SEQ ID NO:3 and SEQ ID NO:4,the complement of SEQ ID NO:3 and the complement of SEQ ID NO:4, SEQ IDNO:7 and SEQ ID NO:8, the complement of SEQ ID NO:7 and the complementof SEQ ID NO:8, SEQ ID NO:11 and SEQ ID NO:12, and the complement of SEQID NO:11 and the complement of SEQ ID NO:12.
 9. A kit comprising: a pairof polynucleotide primers and a pair of labeled polynucleotide probesselected from the group consisting of: (1) the pair of primers are twopolynucleotides comprising at least 12 consecutive nucleotides of SEQ IDNO:1 and SEQ ID NO:2, respectively, and the pair of labeled probes arethe pair of labeled nucleic acids of claim 5 wherein the nucleotidesequence is SEQ ID NO:13; (2) the pair of primers are twopolynucleotides comprising at least 12 consecutive nucleotides of SEQ IDNO:5 and SEQ ID NO:6, respectively, and the pair of labeled probes arethe pair of labeled nucleic acids of claim 5 wherein the nucleotidesequence is SEQ ID NO:14; and (3) the pair of primers are twopolynucleotides comprising at least 12 consecutive nucleotides of SEQ IDNO:9 and SEQ ID NO:10, respectively, and the pair of labeled probes arethe pair of labeled nucleic acids of claim 5 wherein the nucleotidesequence is SEQ ID NO:15.
 10. The kit of claim 9, wherein at least 12consecutive nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:9 and SEQ ID NO: 10 are at least 15 consecutivenucleotides of these sequences.
 11. The kit of claim 9, wherein at least12 consecutive nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:9 and SEQ ID NO:10 are the full length of thesesequences.
 12. A kit comprising: an isolated nucleic acid comprising atleast 12 consecutive nucleotides of SEQ ID NO:1; an isolated nucleicacid comprising at least 12 consecutive nucleotides of SEQ ID NO:2; anisolated nucleic acid comprising at least 12 consecutive nucleotides ofSEQ ID NO:5; an isolated nucleic acid comprising at least 12 consecutivenucleotides of SEQ ID NO:6; an isolated nucleic acid comprising at least12 consecutive nucleotides of SEQ ID NO:9; an isolated nucleic acidcomprising at least 12 consecutive nucleotides of SEQ ID NO:10; a pairof labeled nucleic acids of claim 5 wherein the nucleotide sequence isSEQ ID NO:13; a pair of labeled nucleic acids of claim 5 wherein thenucleotide sequence is SEQ ID NO:14; and a pair of labeled nucleic acidsof claim 5 wherein the nucleotide sequence is SEQ ID NO:15.
 13. Anisolated nucleic acid comprising a nucleotide sequence selected from SEQID NO:13, SEQ ID NO:14 and SEQ ID NO:15.
 14. A method for detecting aSalmonella species, E. coli O157:H7, or Listeria monocytogenescomprising the steps of amplifying a genomic nucleotide sequencecomprising nucleotide 9 to nucleotide 243 of SEQ ID NO:13, nucleotide 7to nucleotide 354 of SEQ ID NO:14, or nucleotide 9 to nucleotide 210 ofSEQ ID NO:15, and detecting an amplification product that contains thegenomic nucleotide sequence.
 15. The method of claim 14, wherein thenucleotide sequence comprises SEQ ID NO:13, SEQ ID NO:14 or SEQ IDNO:15.
 16. A method for detecting a Salmonella species, E. coli O157:H7,or Listeria monocytogenes comprising the steps of amplifying anucleotide sequence of a corresponding species using a pair ofpolynucleotides of claim 9 as PCR primers, and detecting anamplification product.
 17. The method of claim 16, wherein thenucleotide sequence is amplified by real-time PCR and the amplificationproduct is detected by fluorescence resonance energy transfer using apair of labeled polynucleotides of claim
 5. 18. The method of claim 17,wherein the nucleotide sequence is amplified by real-time PCR using apair of polynucleotides of claim
 11. 19. The method of claim 17, whereinthe amplification product is detected by fluorescence resonance energytransfer using a pair of labeled polynucleotides of claim
 8. 20. Themethod of claim 17, wherein the labeled polynucleotides are included andanneal to the amplification product in the amplification step.
 21. Themethod of claim 17, wherein the detection sensitivity is 50 bacteria orfewer.